Therapeutic compositions

ABSTRACT

Compositions comprising ketone bodies and/or their metabolic precursors are provided that are suitable for administration to humans and animals and which have the properties of, inter alia, (i) increasing cardiac efficiency, particularly efficiency in use of glucose, (ii) for providing energy source, particularly in diabetes and insulin resistant states and (iii) treating disorders caused by damage to brain cells, particularly by retarding or preventing brain damage in memory associated brain areas such as found in Alzheimer&#39;s and similar conditions. 
     These compositions may be taken as nutritional aids, for example for athletes, or for the treatment of medical conditions, particularly those associated with poor cardiac efficiency, insulin resistance and neuronal damage. The invention further provides methods of treatment and novel esters and polymers for inclusion in the compositions of the invention.

This application is a continuation of application Ser. No. 10/763,393filed Jan. 26, 2004, which is a continuation of application Ser. No.10/408,667, filed Apr. 8, 2003, which is a continuation of applicationSer. No. 10/153,873, filed May 24, 2002 (abandoned), which is acontinuation of application Ser. No. 09/843,694, filed Apr. 30, 2001,which is a continuation of application Ser. No. 09/397,100, filed Sep.16, 1999 (now U.S. Pat. No. 6,323,237), which is a CIP ofPCT/US98/05072, filed Mar. 17, 1998, which claims benefit of provisionalApplication No. 60/040,853, filed Mar. 17, 1997, the entire contents ofeach of which are hereby incorporated by reference.

THERAPEUTIC COMPOSITIONS

The present invention relates to compositions suitable foradministration to humans and animals which have the properties of, interalia, (i) increasing cardiac efficiency, particularly efficiency in useof glucose, (ii) for providing energy source, particularly in diabetesand insulin resistant states and (iii) treating disorders caused bydamage to brain cells, particularly by retarding or preventing braindamage in memory associated brain areas such as found in Alzheimer's andsimilar conditions. These compositions may be taken as nutritional aids,for example for athletes, or for the treatment of medical conditions,particularly those associated with poor cardiac efficiency, insulinresistance and memory loss. The invention further provides methods oftreatment and novel esters and polymers for inclusion in thecompositions of the invention.

Abnormal elevation of blood sugar occurs not only in insulin deficientand non insulin dependent diabetes but also in a variety of otherdiseases. The hyperglycaemia of diabetes results from an inability tometabolize and the over production of glucose. Both types of diabetesare treated with diet; Type I diabetes almost always requires additionalinsulin, whereas non-insulin dependent diabetes, such as senile onsetdiabetes, may be treated with diet and weight loss, although insulin isincreasingly used to control hyperglycaemia.

Increased sympathetic stimulation or elevated glucagon levels, inaddition to increasing glycogenolysis in liver, also stimulate freefatty acid release from adipocytes. After acute myocardial infarction orduring heart failure, increased sympathetic nervous-activity oradministration of sympathomimetics accelerate glycogenolysis, decreaserelease of insulin from P cells of the pancreas and cause relativeinsulin resistance. While the importance of diet, or substrateavailability, is taken as a given in the treatment of diabetes, thecritical effects of substrate choice in insulin resistant states has notbeen widely appreciated or applied in clinical practice. Insteadcontemporary interest has focused upon the complex signalling cascadewhich follows the binding of insulin to its receptor. This increasinglycomplex cascade of messages involving protein tyrosine kinases andphosphatases, inositol and other phospholipids, while holding promisefor the ultimate understanding of non-insulin dependent diabetes, hasyet to provide significant new therapies for either diabetes or insulinresistance.

Leaving aside the longer term effects of insulin on growth, the acutemetabolic effects of insulin have been thought to be accounted for byaction at three major enzymatic steps in the conversion of glucose toCO₂. Firstly insulin promotes the translocation of the glucosetransporter, Glut4, from endoplasmic reticular to plasma membranes, thusincreasing the transport of glucose from the extra to intracellularphase.(see refs. 1 and 2). Secondly, insulin increases the accumulationof glycogen. This has been attributed to dephosphorylation of glycogensynthase (3) by protein phosphatase 1. Thirdly, insulin stimulates theactivity of mitochondrial pyruvate dehydrogenase multi-enzyme complex (4and 5) through dephosphorylation by a Ca²⁺ sensitive (6)intramitochondrial protein phosphosphatase.

An important, but poorly understood effect of insulin is its use incardiac disease where in combination with glucose, potassium chlorideand GIK, it improved electrocardiographic abnormalities accompanyingmyocardial infarction (7 and 8), and improved cardiac performance afterpost pump stunning (9). This treatment has been advocated recently for anumber of other serious cardiac diseases (10 and 11). The beneficialeffects of GIK infusion have been attributed to its ability to decreasefree fatty acid release and improve membrane stability (12). However,other more recent work suggests more fundamental reasons. In heart cellsthat are anoxic, glucose is the only fuel capable of providing the ATPnecessary to maintain viability (13).

Administration of glucose plus insulin would increase the availabilityof intracellular glucose providing a source of ATP production in theabsence of O₂. While this would explain certain beneficial effects, itwould not account for the correction of EKG abnormalities nor theimproved cardiac index in hearts treated with GIK because electricalactivity and cardiac work requires actively respiring cardiac cells, notones which are totally anoxic and therefore without electrical activityor the ability to perform mechanical work.

Understanding the enzymatic sites of insulin's action does not, byitself, define the effects of insulin deficiency upon the cellularmetabolism or physiological function. How insulin acts at this largerlevel can best be understood by looking at the way nature deals withinsulin deficiency. The natural compensation for decreased insulinduring fasting is the accelerated hepatic conversion of the free fattyacids to the ketone bodies raising blood D-β-hydroxybutyrate andacetoacetate to about 6 mM. At these levels, ketones, rather thanglucose, become the substrate for most organs, including even the brain(14). Although mild ketosis is the normal response to decreased insulin,physicians fear ketone bodies because their massive overproduction canbe life threatening in diabetic ketoacidosis.

The present inventor has previously compared the effect of physiologicallevels of ketone bodies to the metabolic and physiological effects ofinsulin, particularly comparing the insulin deficient working rat heartperfused with glucose alone, to hearts to which was added either 4 mMD-β-hydroxybutyrate/1 mM acetoacetate, saturating doses of insulin orthe combination and has shown how provision of simple substrates canmimic the effects of insulin in changing the concentrations of theintermediates of both glycolysis and the TCA cycle and therebycontrolling the flux of glucose in this very specialised tissue. Inaddition he has determined that a primary but previously unrecognizedeffect of insulin or a ratio of ketones is to alter mitochondrial redoxstates in such a way so as to increase the ΔG_(ATPhydrolysis) and withthat, the gradients of inorganic ions between the various cellularphases and the physiological performance of heart.

The present application teaches that such ketone bodies can also providea therapeutic approach to the treatment of insulin resistance where thenormal insulin signalling pathway is disordered and in conditions wherethe efficiency of cardiac hydraulic work is decreased for metabolicreasons. The inventor has determined that use of ketone bodies has greatadvantage over use of insulin itself for reasons that will becomeevident from the description below, not least of these being theelimination of carbohydrate intake control otherwise necessary.

The present application further addresses the problem ofneurodegenerative diseases, particularly disease where neurons aresubject to neurotoxic effects of pathogenic agents such as proteinplaques and further provides compositions for use in treating these andthe aforesaid disorders.

Alzheimer's disease is a genetically heterogeneous group ofprogressively fatal neurological diseases characterized pathologicallyby accumulation of amyloid plaques in brain and clinically by impairmentof recent memory leading to dementia and death. In addition to the casesof Alzheimer's disease linked to genetic causes, sporadic cases, withoutan apparent family history of the disease, also occur. For examplepathological changes characteristic of Alzheimer's disease occur afterhead trauma (73) or after inflammatory diseases stimulating productionof the cytokine interleukin-1 (97).

The early symptom of the disease is loss of recent memory associatedwith impairment and death of cell in the hippocampus accounting for theearly impairment of recent memory. Measurement of the hippocampalvolumes using magnetic resonance imaging (MRI) shows that atrophy ofhippocampus occurs prior to the clinical onset of memory loss andprogresses with a loss of volume of about 8% per year during the 2 yearsover which symptoms first appeared (70).

The diagnosis of Alzheimer's disease is made clinically by thisimpairment in recent memory, associated with lesions in the hippocampalportion of the temporal lobe. Neuropathologically, the diagnosis dependsupon the finding of neurofibrillatory tangles within the cells, amyloidor senile plaques in the extracellular space and loss of neuronal number(61). The neurofibrillatory tangles are comprised of pairedhyperphosphorylated tau protein, whose usual function in the cell, whennot phosphorylated, is to bind to and stabilize tubulin in its formationof microtubules within the cell. Hyperphosphorylation of tau iscatalysed by glycogen synthase kinase 3β, among other kinases anddephosphorylated by protein phosphatase 2A-1, 2B or 1(108).

However, there is not necessarily a clear, bright line between thepathological brain changes and the memory deficits which occurprematurely in Alzheimer's disease and the pathological changes in brainanatomy and memory function which are found in the “normal” agingpopulation. Rather the difference is a quantitative one dependent uponrate (94). Such changes in memory function in the normal aged are alsoaccompanied by a decreased glucose tolerance signifying an inability tometabolize glucose. In such situations, treatments aimed at rectifyingthe pathophysiological processes of Alzheimer's disease, would beexpected to be applicable to the correction of the metabolic effectsassociated with normal aging.

While Alzheimer's disease of the familial or the sporadic type is themajor dementia found in the aging population, other types of dementiaare also found. These include but are not limited to: thefronto-temporal degeneration associated with Pick's disease, vasculardementia, senile dementia of Lewy body type, dementia of Parkinsonismwith frontal atrophy, progressive supranuclear palsy and corticobasaldegeneration and Downs syndrome associated Alzheimers'. Plaque formationis also seen in the spongiform encephalopathies such as CJD, scrapie andBSE. The present invention is directed to treatment of suchneurodegenerative diseases, particularly those involving neurotoxicprotein plaques, eg. amyloid plaques.

Many of these aforesaid apparently unrelated conditions have thehyperphosphorylated tau proteins found in Alzheimer's disease (69),opening up the possibility that the same kinase which phosphorylated tauwould also phosphorylate the PDH complex producing a similar deficiencyin mitochondrial energy production and acetyl choline synthesis found inAlzheimer's disease but involving other brain regions. The presentinventor has determined that in this respect treatments applicable toAlzheimer's disease might be applied to these diseases as well. Inaddition, the inventor has determined that such treatment will also beapplicable to peripheral neurological wasting diseases, such asmyasthenia gravis and muscular dystrophy.

At present there is no effective treatment for Alzheimer's disease.Research efforts are focused on defining its genetic cause but to datethere has been no succesful gene therapy. Genetic studies have linkedAlzheimer's disease with Mongolism and in its early onset form to locuson chromosome 21 causing accumulation of amyloid precursor protein (APP)(73), a transmembrane glycoprotein existing in 8 isoforms. Numerousfragments of this protein are derived by proteolysis and the plaquescharacteristic of Alzheimer's disease have been shown to containaccumulation of the oligomer of β amyloid protein (A β₁₋₄₂). An earlyonset autosomally dominant form of Alzheimer's disease has also beenrelated to a presenilin 1 locus on chromosome 14.

A late onset form of Alzheimer's disease is associated with the type 4allele of apolipoprotein E (69,98) on chromosome 19, although otherworkers suggest that this apparent correlation may be related instead ofα 1 antichymotrypsin locus instead (100). All transgenic mice expressingincreased amounts of amyloid precursor protein over 18 months of ageshowed hippocampal degeneration with many of the pathologicalcharacteristics of Alzheimer's disease (90).

The current status of knowledge on the defective genes and gene productsin Alzheimer's disease has recently been summarized (Table 1 of ref.96).

Chro- mosome Gene Defect Age of Onset Aβ Phenotype 21 βAPP mutations50's Production of total Aβ peptides of Aβ₁₋₄₂ 19 apoE4 polymorphism60's or > density of Aβ plaques and vascular deposits 14 Presenilin 1mutations 40's & 50's production of Aβ₁₋₄₂ 1 Presenilin 2 mutations 50'sproduction of Aβ₁₋₄₂

It is clear from the above table that the common phenotype associatedwith the genetic forms of Alzheimer's disease is the accumulation of theamyloid peptide Aβ₁₋₄₂ (96). It is this Aβ₁₋₄₂ which inactivates PDHthus impairing mitochondrial energy and citrate production in normallyobligate glucose consuming tissue (95) and at the same time impairingsynthesis of the critical neurotransmitter, acetyl choline (67,68). Theapplication of Aβ₁₋₄₂ to neuronal cells is associated with thedownregulation of the anti-apototic protein bcl-1 and increases levelsof bax, a protein known to be associated with cell death (92). Inaddition to amyloid plaques comprised of Aβ₁₋₄₂, neurofibrillatorytangles comprised of hyperphosphorylated tau protein, and decreasedbrain acetyl choline levels, cell death is the fourth pathologicalcharacteristic of Alzheimer's disease. These pathologicalcharacteristics can be related, at least in part, to excess Aβ₁₋₄₂ andits inhibition of PDH.

Modest clinical improvement in symptoms can occur by treatment withacetyl choline esterase inhibitors (57), presumably by increasingcholinergic efferents originating in the septal nuclei and traversingBroca's diagonal band to hippocampus in the anterior portion of thelimbic system of brain. However the progress in the molecular biology ofAlzheimer's disease has caused the search for new therapies toconcentrate upon four major areas (96): (i) protease inhibitors thatpartially decrease the activity of the enzymes (β and γ secretase) thatcleave Aβ (β amyloid fragments) from βAPP (β amyloid precursorproteins); (ii) compounds that bind to extracellular Aβ that prevent itscytotoxic effects; (iii) brain specific anti-inflammatory drugs thatblock the microglial (brain macrophages) activation, cytokine release,and acute phase response that occur in affected brain regions; and (iv)compounds such as antioxidants, neuronal calcium channel blocks, orantiapoptotic agents that interfere with the mechanisms of Aβ triggeredneurotoxicity.

The therapy which the present inventor now proposes differs from thefour approaches listed above in that it bypasses the block in metabolicenergy production resulting from inhibition of PDH by Aβ₁₋₄₂ byadministering ketone bodies or their precursors. Neuronal cells arecapable of metabolizing such compounds even in the presence of adeficiency of glucose, the normal energy substrate for brain (63).Because ketones can increase the ΔG of ATP hydrolysis, the gradients ofboth intracellular Na⁺ and Ca²⁺ will be increased, preventing cell deathassociated with increased intracellular Ca2+. Furthermore, the increasein citrate generation by the Krebs cycle will provide, when translocatedinto cytoplasm, a source of cytoplasmic acetyl CoA required to remedythe deficiency of acetyl choline characteristic of Alzheimer's brains.

The elevation of blood ketones necessary to correct these metabolicdefects can be accomplished by parenteral, enteral means or dietarymeans and does not require the administration of potentially toxicpharmacological agents.

There has been long experience with ketogenic diets in children treatedfor epilepsy. Such diets are however unsuitable for use in adults due toadverse efects on the circulatory system. The present inventionsapplication of ketone bodies should provide all the therapeutic effectsof such diet, which is not itself found to be toxic in children, withnone of the side effects that render it unused adults. Furthermore, theinventor has determined that with the correction of the aforesaidmetabolic defects, cytokine responses and the increase in apoptoticpeptides in degenerating cells will decrease due to the increase inneuronal cell energy status and the increased trophic stimulationresulting from increased acetyl choline synthesis.

Since the priority date of this application, EP 0780123 A1 has beenpublished which relates to use of acetoacetate, β-hydroxybutyrate,monhydric, dihydric or trihydric alcohol esters of these or oligomers ofβ-hydroxybutyrate for suppressing cerebral edema, protecting cerebralfunction, rectifying cerebral energy metabolism and reducing the extentof cerebral infarction. It should be noted however, that it has beenknown since 1979 that sodium hydroxybutyrate increases cerebralcirculation and regional vasomotor reflexes by up to 40% (Biull. Eksp.Biol. Med Vol 88 11, pp 555-557). The treatment that the presentinventor now provides goes beyond such effects on circulation as itprovides treatment for cells that are unable to function due toneurodegeneration, eg caused by neurotoxic agents such as peptides andproteins, and genetic abnormality. The treatment involves action ofketone bodies on the cells themselves and not the flow of blood to them.

In reducing this invention to practice the inventor has furtherdetermined that ketone bodies, provided by direct adminsitration or byadministration of their metabolic precursors in amounts sufficient toraise total blood ketone body concentration to elevated levels result inmore than simple maintenance of cell viability but actually improve cellfunction and growth beyond that of normal, ie. control levels in amanner unrelated to blood flow or nutrition. In this respect theinvention further provides use of ketone bodies as nerve stimulantfactors, ie. nerve growth factors and factors capable of stimulatingenhanced neuronal function, such as increase of metabolic rateand.increase of extent of functional features such as axons anddendrites. This aspect of the present invention offers a mechanism forimprovement of neuronal function as well as mere retardation ofdegredation.

The recent work of Hoshi and collaborators (77, 78) strongly suggeststhat a part of the amyloid protein whose accumulation is the hallmark ofAlzheimer's disease, Aβ₁₋₄₂, acts as a mitochondrial histidine proteinkinase which phosphorylates and inactivates the pyruvate dehydrogenasemultienzyme complex. The PDH complex is a mitochondrial enzymeresponsible for the generation of acetyl CoA and NADH from the pyruvateproduced by glycolysis within the cytoplasm. The mitochondrial acetylCoA formed condenses with oxaloacetate to start the Krebs TCA cyclecompletely combusting pyruvate to CO₂ while providing the mitochondriawith the reducing power which becomes the substrate for the electrontransport system through which the energy required for mitochondrial ATPsynthesis is generated. PDH thus stands at the crossroads of the twomajor energy producing pathways of the cell, glycolysis and the Krebscycle, and clearly serves a critical function in living cells.

There are two major consequences of the inhibition of PDH. Firstly, inneuronal tissues, which under normal metabolic conditions are totallydependent upon glucose for energy production, inhibition of PDH resultsin a lowered efficiency of energy production, a lowered energy ofhydrolysis of ATP, a decrease in both acetyl CoA and the metabolites ofthe first ⅓ of the TCA cycle and a deficiency of mitochondrial NADH(95). A decrease in the energy of ATP hydrolysis leads to increasedintracellular Na⁺ and Ca²⁺, loss of cellular K⁺ and ultimately celldeath (86). Hippocampal cells, critical for the fixation of recentmemories, are particularly sensitive to a number of forms of injury, andthe death of these cells is the hallmark both clinically andpathologically of Alzheimer's disease.

A second major consequence of PDH inhibition is a deficiency ofmitochondrial citrate (95). Citrate, or one of its metabolites, isexported to the cytoplasm from mitochondria where it is converted tocytosolic acetyl CoA by ATP citrate lyase (EC 4.1.3.8) in the reaction:

citrate³⁻+ATP⁴⁻+CoASH>acetyl CoA+oxaloacetate²⁻+ADP³⁻+HPO₄ ²⁻

The acetyl CoA then combines with choline through the action of cholineacetyl transferase

(EC 2.3.1.6) to form acetyl choline in the reaction:

choline⁺+acetyl CoA>CoASH+acetyl choline⁺

Neuronal culture of septal cells exposed to 1 μm Aβ₁₋₄₂ for 24 hoursshowed a decrease in acetyl choline production of over five fold (78)with no decrease in the activity of choline acetyl transferase. Theinferred cause of this decreased production was a deficiency of acetylCoA due to inhibition of the PDH complex caused by activation of theTPKI/GSK-3β protein kinase and subsequent phosphorylation of PDH (77).

As explained above isolated working hearts perfused with 10 mM glucosealone without insulin are inefficient and have impaired mitochondrialenergy production. This defect in cellular energy production can becompletely reversed by the provision of a physiological ratio of ketonebodies consisting of 4 mM D-β hydroxybutyrate and 1 mM acetoacetate(95). Brain was thought to be capable of using only glucose as itsmetabolic energy source and to be insensitive to the actions of insulin.However, in a remarkable clinical study performed in 1967, George Cahilland his collaborators (47) showed that up to 60% of the brain's need formetabolic energy could be met by ketone bodies in obese patientsundergoing prolonged fasting. Even more remarkably, Cahill showed thatadministration of insulin to these patients in doses sufficient to droptheir blood sugar from 4 to under 2 mM was associated with no impairmentof mental functions in these patients whose blood D-β hydroxybutyratewas 5.5 mM and acetoacetate 2 mM (see FIG. 3 from ref 63). Clearly, whenketone bodies are present in the blood at levels above 5 mM, they areable to substitute for the brain's usual need for glucose and abolishthe hypoglycemic symptoms expected at blood glucose levels of 1.5 mM

Ketone body utilization in brain is limited by the transport, withlesser utilization occurring in the basal ganglion at blood levels below1 mM (76). However, at levels of 7.5 mM achieved in normal man byprolonged fasting, the rate of ketone body entry into brain issufficient to take over the majority of cerebral energy needs and toprevent hypoglycemic symptoms, even in the face of blood sugar levelswhich would normally cause convulsions or coma (63) .

It is the inventors hypothesis that in Alzheimer's disease, where thereis a block at PDH which prevents the normal energy production fromglucose, if one can provide elevated, eg. normal fasting levels ofketones, one can bypass the PDH blockade present in these patientsthereby preventing cell death due to energy depletion or lack ofcholinergic stimulation and thus slow the progression of the memory lossand dementia.

Furthermore, utilising the nerve growth/stimulatory effects of theketone bodies, particularly D-β-hydroxybutyrate or a physiological ratioof this with acetoacetate, cells that are still viable can be caused toimprove beyond the state to which they have degenerated and accordinglysome improvement of function will be seen in patients.

In fed animals and in man the liver content, which is essentially thatof blood, of acetoacetate is very low at 0.09 mM and D-β hydroxybutyrateis 0.123 mM but rises after a 48 hour fast to 0.65 mM acetoacetate and1.8 mM D-β hydroxybutyrate (84). The ketone bodies rise in starvationbecause the fall in insulin decreases the re-esterification of fattyacids to triglyceride in adipose tissue causing the release of freefatty acids into the blood stream. The released free fatty acids canthen be taken up and used as a source of energy by muscle, heart, kidneyand liver in the process of β oxidation. Liver, however, has thecapacity to convert the free fatty acids to a metabolic fuel, ketones,for use by extrahepatic organs, including the brain, as an alternativeto glucose during periods of fasting. The hepatic synthesis of ketonebodies occurs from mitochondrial acetyl CoA generated during theβ-oxidation of fatty acids by liver in the following set of reactions:

Once made in the liver, ketone bodies are transported out of the liverinto the blood stream by the monocarboxylate —H′ co-transporter (20) bythe following reaction:

The ketone bodies enter extra-hepatic tissues on the same carrier, whereother monocarboxylates can act as competitive inhibitors.Unphysiological isomers such as D-lactate or L-β-hydroxybutyrate canalso act as competitive inhibitors to ketone body transport. Sinceketone body transport across the blood brain barrier is the limitingfactor to ketone body utilization in brain (76) every effort should bemade to keep the blood concentration of these unphysiologicalenantiomers at low levels during ketogenic therapy. When blood ketonebody concentrations are elevated to levels found in starvation, heart,muscle, kidney and brain utilize ketone bodies as the preferred energysubstrate:

The present inventor has thus determined that the mitochondrial acetylCoA from ketone bodies can thus replace the acetyl CoA deficiency whichoccurs during inhibition of PDH multienzyme complex in tissues dependentupon the metabolism of glucose for their supply of metabolic energy. Themitochondrial citrate supplied can also be transported to cytoplasm bythe tri or dicarboxcylic acid transporter where it can be converted tocytoplasmic acetyl CoA required for the synthesis of acetyl choline. Thereactions of the Krebs cycle are shown in FIG. 3 to help illustratethese concepts further.

The liver cannot utilize ketone bodies because it lacks the 3 OxoacidCoA transferase necessary for the formation of acetoacetyl CoA. Ketonebodies, in contrast to free fatty acids, cannot produce acetyl CoA inliver. Since acetyl CoA is the essential precursor of fatty acidsynthesis through malonyl CoA and cholesterol synthesis throughcytosolic HMG CoA, ketone bodies cannot result in either increased fattyacid or cholesterol synthesis in liver, which usually accounts for overhalf of the bodies synthesis of these two potentially pathogenicmaterials. Liver is sensitive to the ratio ofactoacetate/D-β-hydroxybutyrate presented to it and will alter itsmitochondrial free [NAD⁺]/[NADH], because of the near equilibriumestablished by β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) (55)

The easiest way to increase blood ketones is starvation. On prolongedfasting blood ketones reach levels of 7.5 mM (62, 63). However, thisoption is not available on a long term basis, since death routinelyoccurs after a 60 day fast.

The ketogenic diet, comprised mainly of lipid, has been used since 1921for the treatment of epilepsy in children, particularly myoclonic andakinetic seizures (109) and has proven effective in cases refractory tousual pharmacological means (71). Either oral or parenteraladministration of free fatty acids or triglycerides can increase bloodketones, provided carbohydrate and insulin are low to preventre-esterification in adipose tissue. Rats fed diets comprised of 70%corn oil, 20% casein hydrolysate, 5% cellulose, 5% McCollums saltmixture, develop blood ketones of about 2 mM. Substitution of lard forcorn oil raises blood ketones to almost 5 mM (Veech, unpublished).

An example of a traditional 1500/day calorie ketogenic diet recommendedby the Marriott Corp. Health Care Services, Pediatric Diet Manual,Revised August 1987 as suitable for a 4-6 year old epileptic childcontained from 3:1 to 4:1 g of fat for each g of combined carbohydrateand protein. At each of 3 meals the patient must eat 48 to 50 g fat,only 6 g protein and 10 to 6.5 g carbohydrate. In practice this meansthat at each meal the child must eat 32 g of margarine per day (about ¼stick) and drink 92 g of heavy cream (about 100 ml), comprised mainly asmedium chain length triglycerides.

An example of a diet achieving a 3:1 ratio of fat to combinedcarbohydrate and protein is given in Table 1 below.

TABLE 1 Sample 1500 calorie diet to achieve 3:1 lipid to carbohydrate +protein diet Amount (g) Fat (g) Protein (g) CHO (g) Breakfast Egg 32 4 4apple juice 70 7 margarine 11 10 heavy cream 92 34 2 3 Total Breakfast48 6 10 Lunch lean beef 12 1.75 3.5 cooked carrots 45 0.6 3 canned pears40 4 margarine 14 12.5 heavy cream 92 34 2 3 Total Lunch 48.25 6.1 10Supper Frankfurter 22.5 6 3 Cooked broccoli 50 1 2 Watermelon 75 5Margarine 8 7.5 Heavy cream 92 34 2 3 Total Supper 47.5 6 10 Daily Total143.75 18.1 30

In general the levels of ketone bodies achieved on such diets are about2 mM D-β hydroxybutyrate and 1 mM acetoacetate while the levels of freefatty acids about 1 mM. Other variations of composition have been triedincluding medium chain length triglycerides. In general compliance withsuch restricted diets has been poor because of their unpalatability(56). High lipid, low carbohydrate diets also have been tried astherapeutic agents in cancer patients to reduce glucose availability totumors (88) as weight reducing diets in patients with and withoutdiabetes (74, 112) to improve exercise tolerance (83).

The limitation of diets which rely upon lipid to raise blood ketones toneurologically effective levels are many. Firstly, levels of ketonebodies on lipid based diets tend to be below 3 mM, significantly lowerthan the level of 7.5 mM achieved in normal obese humans duringprolonged fasting. Secondly, unauthorized ingestion of carbohydrateincreases insulin secretion and causes a rapid decrease in the hepaticconversion of free fatty acids to ketones with a consequent drop inblood ketones and the diversion of lipid to esterified to triglyceridesby adipose tissue. Many anecdotal reports relate the resumption ofseizures in children who “broke their diet with birthday cake”. Thirdlythe unpalatability and the necessity to avoid carbohydrate to sustainhigh ketone body levels makes such high lipid diets difficult to use inadults in an out patient setting, particularly in societies wheretraditionally high intake of refined sugars, bread, pasta, rice andpotatoes occurs. In practice, the traditional high ketone diet cannot beenforced in patients, other than children beyond the age where all foodis prepared at home under strict supervision. Fourthly, ingestion ofsuch large amounts of lipid in the adult population would lead tosignificant hypertriglyceridemia with its pathological sequelae ofincreased vascular disease and sporadic hepatic and pancreatic disease,and therefore could not be prescribed on medical grounds. Ingestion ofhigh lipid, low carbohydrate diets were popular in the 1970s for weightreduction in the face of high caloric intake, provided that carbohydrateintake was low. However, because of the increased awareness of therelationship of elevated blood lipids to atherosclerosis the popularityof this diet dropped abruptly.

Supplementing a liquid diet with 47% of its caloric content with eitherglucose or racemic 1,3 butandiol caused the blood ketone concentrationto rise about 10 fold to 0.98 mM D-β hydroxybutyrate and 0.33 mMacetoacetate (107). These values are slightly less than obtainednormally in a 48 hour fast and far below the levels of 7.5 mM obtainedin fasting man. Racemic 1,3 butandiol is converted by liver toacetoacetate and both the unnatural L-β and the natural D-βhydroxybutyrate (respectively (S) 3-hydroxybutanoate and (R)3-hydroxybutanoate). Although racemic 1,3 butandiol has been extensivelystudied as a cheap caloric source in animal food and has even been usedexperimentally in human diets (81, 101) the production of the unnaturalL-isomer is likely in the long run to produce significant toxicity ashas been shown for the human use of the unnatural D-lactate (64). Onedisadvantage of administering the unnatural L isomer is that it competesfor transport with the natural D-β hydroxybutyrate. Thus provision ofthe (R) 1,3 butandiol as a precursor of ketone bodies is one possibilitythat avoids unnecessary administration or production of the unnaturalisomer.

The mono and diester of racemic 1,3 butandiol have been suggested as asource of calories and tested in pigs (67). Oral administration of abolus of a diet containing 30% of calories as the esters produced briefpeaks blood ketones to 5 mM. However, the use of racemic 1,3 butandiolwith its production of the abnormal (S) 3-hydroxybutanoate is not to berecommended for the reasons stated above.

While use of racemic 1,3 butandiol in such formulations is notrecommended, the esters of (R) 1,3 butandiol can be used, either aloneor as the acetoacetate ester. (R) 1,3 butandiol may easily besynthesized by reduction of the monomeric D-β hydroxybutyrate, with forexample LiAlH₄. (R) 1,3 butandiol is subject to being oxidized in theliver to form D-β hydroxybutyrate without marked distortion of thehepatic redox state. Studies in rats have shown that feeding racemic 1,3butandiol caused liver cytosolic [NAD′]/[NADH] to decrease from 1500 toabout 1000 (87). By comparison, administration of ethanol reduceshepatic [NAD-]/[NADH] to around 200 (106).

Acetoacetate, when freshly prepared, can be used in infusion solutionswhere it can be given in physiologically normal ratios to optimum effect(95). Because of manufacturing requirements which currently require longshelf life and heat sterilized fluids, acetoacetate has frequently beengiven in the form of an ester. This has been done to increase its shelflife and increase its stability to heat during sterilization. In theblood stream, esterase activity has been estimated to be about 0.1mmol/min/ml and in liver about 15 mmol/min/g (68). In addition to esterscombining 1,3 butandiol and acetoacetate there has also been extensivestudy of glycerol esters of acetoacetate in parenteral (59) and enteralnutrition (82). Such preparations were reported to decrease gut atrophy,due to the high uptake of acetoacetate by gut cells and to be useful intreatment of burns (85).

However, neither 1,3 butandiol, which forms acetoacetate, nor glycerol,which is a precursor of glucose, is part of the normal redox couple, D-βhydroxybutyrate/acetoacetate. For the present invention, under optimumconditions, a physiological ratio of ketones should be given. If it isnot, in the whole animal, the liver will adjust the ratio of ketones inaccordance with its own mitochondrial free [NAD⁺]/[NADH]. If an abnormalratio of ketones is given pathological consequences are a distinctpossibility. In the working heart, perfusion with acetoacetate as solesubstrate, rapidly induces heart failure (99) in contrast to rat heartsperfused with a mixture of glucose, acetoacetate and D-βhydroxybutyrate, where cardiac efficiency was increased by aphysiological ratio of ketone bodies (95).

The best exogenous source of ketone bodies, which do not requireingestion of large amounts of lipid nor the use of material whichproduce the physiologically incompatible isomers L-β-hydroxybutyratewould be ketone bodies themselves. However the present invention alsoprovides alternatives for administration in therapy.

A first alternative are polyesters of D-β-hydroxybutyrate. Naturalpolyesters of D-β-hydroxybutyrate are sold as articles of commerce atpolymers of 530,000 MW from Alcaligenes eutrophus (Sigma Chemical Co.St. Louis) or as 250,000 MW polymers for sugar beets (Fluka,Switzerland). The bacteria produce the polymer as a source of storednutrient. The fermentation of these polymers by bacteria was developedin the 1970s by ICI in the UK and Solvay et Cie in Belgium, as apotentially biodegradable plastic for tampon covers and other uses. Thesystem responsible for the synthesis of the poly D-β-hydroxybutyrate hasnow been cloned and variations in the composition of the polymerproduced, based on the substrates given to the bacteria demonstrated.However, these polymers failed to be able to compete with petroleumbased plastics. Nevertheless the genes responsible for the synthesis ofpolyalkanoates has been cloned and expressed in a number ofmicro-organisms (93, 102, 113) allowing for production of this materialin a variety of organisms under extremely variable conditions.

Poly D-β-hydroxybutyrate comes in a number of forms from differentbiological sources as an insoluble white powder with little taste and noodour and is suitable for incorporation into compositions for oral orother means of administration. Esterases capable of breaking the esterbonds of this material are ubiquitous in plasma and most cells. Thesepolymer are also easily split by alkaline hydrolysis in vitro to make aseries of polymers culminating in the production of the monomer of MW104, which is transported from gut to portal vein by the normalmonocarboxylate transporter. Alternatively acid hydrolysis may becarried out using the published method referred to in the Flukapromotional material.

Preferred forms of D-β-hydroxybutyrate polymer are oligomers of thatketone body designed to be readily digestable and/or metabolised byhumans or animals. These preferably are of 2 to 100 repeats long,typically 2 to 20 and most conveniently from 3 to 10 repeats long. Itwill be realised that mixtures of such oligomers may be employed withadvantage that a range of uptake characteristics might be obtained.

Particularly preferred are cyclic oligomers of D-β-hydroxybutyrate,known as oligolides, having formula

-   -   where n is an integer of 1 or more    -   or a complex thereof with one or more cations or a salt thereof

Preferred cations are sodium, potassium, magnesium and calcium. Suchcations are typically balanced by a physiologically acceptablecounter-anion such that a salt is provided.

Examples of typical physiologically acceptable salts will be selectedfrom sodium, potassium, magnesium, L-Lysine and L-arginine or eg. morecomplex salts such as those of methyl glucamine salts

Preferably n is an integer from 1 to 200, more preferably from 1 to 20,most preferably from 1 to 10 and particularly conveniently is 1, ie. (R,R, R)-4,8,12-trimethyl-1, 5,9-trioxadodeca-2,6,10-trione, 2, 3, 4 or 5.

Cyclic oligomers for use in the invention may be provided, inter alia,by methods described by Seebach et al. Helvetia Chimica Acta Vol 71(1988) pages 155-167, and Seebach et al. Helvetia Chimica Acta, Vol 77(1994) pages 2007 to 2033. For some circumstances such cyclic oligomersof 5 to 7 or more (R)-3-hydroxybutyrate units may be preferred as theymay be more easily broken down in vivo. The methods of synthesis of thecompounds described therein are incorporated herein by reference.

In preferred forms of all of the aspects of the invention, where theoligomer of of D-β-hydroxybutyrate does not include acetoacetyl groupsit is optionally and preferably administered together with aphysiological ratio of acetoacetate or a metabolic precursor ofacetoacetate.

Once the monomer is in the blood stream, and since liver is incapable ofmetabolizing ketone bodies but can only alter the ratio ofD-β-hydroxybutyrate/acetoacetate, the ketone bodies are transported toextrahepatic tissues where they can be utilized. The blood levels ofketones achieved are not be subject to variation caused by noncompliantingestion of carbohydrate, as is the case with the present ketogenicdiet. Rather, they would simply be an additive to the normal diet, givenin sufficient amounts to produce a sustained blood level, typically ofbetween 0.3 to 20 mM, more preferably 2 to 7.5 mM, over a 24 hourperiod, depending upon the condition being treated. In the case ofresistant childhood epilepsy, blood levels of 2 mM are currently thoughtto be sufficient. In the case of Alzheimer's disease, attempts could bemade to keep levels at 7.5 mM achieved in the fasting man studies, in aneffort to provide alternative energy and acetyl CoA supplies to braintissue in Alzheimer's patients where PDH capacity is impaired because ofexcess amounts of Aβ₁₋₄₂ amyloid peptide (77, 78).

The determination by the inventor that D-β-hydroxybutyrate and itsmixtures with acetoactetate act as a nerve stimulant, eg. nerve growthstimulant and/or stimulant of axon and dendritic growth, opens up theoption of raising ketone body levels to lesser degrees than requirednutritionally in order to treat neurodegeneration.

Compositions of the invention are preferably sterile and pyrogen free,particularly endotoxin free. Secondly, they are preferably formulated insuch a way that they can be palatable when given as an additive to anormal diet to improve compliance of the patients in taking thesupplements. The oligomers and polymers are generally taste and smellfree. Formulations of D-β-hydroxybutyrate and its mixtures withacetoacetate may be coated with masking agents or may be targeted at theintestine by enterically coating them or otherwise encapsulating them asis well understood in the pharmaceuticals art.

Since ketone bodies contain about 6 calories/g, there is preferably acompensatory decrease in the amounts of the other nutrients taken toavoid obesity.

Particular advantages of using the ketone bodies or precursors such aspoly or oligo-D-β-hydroxybutyrate or acetoacetate esters are:

1) they can be eaten with a normal dietary load of carbohydrate withoutimpairing its effects,

2) they will not raise blood VLDL, as with current cream and margarinecontaining diets, thus eliminating the risk of accelerated vasculardisease, fatty liver and pancreatitis,

3) they will have a wider range of use in a greater variety of patients,including: type II diabetes to prevent hypoglycemic seizures and coma,in Alzheimer's disease and other neurodegenerative states to preventdeath of nerve cells eg. hippocampal cells, and in refractory epilepsydue to either decreases in cerebral glucose transporters, defects inglycolysis, or so called

Leigh's syndromes with congenital defects in PDH.

The second group of particular alternatives are acetoacetate esters ofD-β-hydroxybutyrate. Esters which provide a physiological ratio ofacetoacetate to D-β-hydroxybutyrate are preferred eg. from 1:1 to 1:20,more preferably from 1:1 to 1:10. The tetramer of D-β-hydroxybutyratewith a terminal acetoacetate residue is particularly preferred. Suchmaterials have the added virtue of having a physiological ratio ofD-β-hydroxybutyrate/acetoacetate moieties, thus removing the burden onliver of having to adjust the redox state of the administered nutrientwithout inducing abnormal reduction of hepatic [NAD⁺]/[NADH] as occurswith excessive alcohol consumption. The polymeric esters, depending upontheir length, have decreasing water solubility, but are heat stable.Such polymers can for example be used in oral and parenteral use inemulsions, whereas acetoacetate, in the unesterified state, is lesspreferred as it is subject to spontaneous decarboxylation to acetonewith a half time at room temperature of about 30 days.

Examples of poly D-β-hydroxybutyrate or terminally oxidized polyD-β-hydroxybutyrate esters useable as ketone body precursors are givenbelow.

Poly (R) 3-Hydroxybutyric Acid

Oxidized Poly (R) 3-Hydroxybutyric Acid

In each case n is selected such that the polymer or oligomer is readilymetabolised on administration to a human or animal body to provideelevated ketone body levels in blood. Preferred values of n areinetegers of 0 to 1,000, more preferably 0 to 200, still more preferably1 to 50 most preferably 1 to 20 particularly conveniently being from 3to 5.

A number of variations of this material, including the polyesterD-β-hydroxybutyrate itself can also be tried for suitable manufacturingcharacteristics. The material is a tasteless white powder. After partialalkaline hydrolysis, a mixture of varying chain length polymers would beprovided, which would tend to smooth gut absorption and maintain highsustained levels of ketone over a 24 hour period.

Treatment may comprise provision of a significant portion of the caloricintake of patients with the D-β-hydroxybutyrate polyester formulated togive retarded release, so as to maintain blood ketones in the elevatedrange, eg. 0.5 to 20 mM, preferably 2-7.5 mM, range over a 24 hourperiod. Release of the ketone bodies into the blood may be restricted byapplication of a variety of techniques such as microencapsulation,adsorption and the like which is currently practised in the oraladministration of a number of pharmaceutical agents. Enetrically coatedforms targeting delivery post stomach may be particularly used where thematerial does not require hydrolysis in acid environment. Where somesuch hydrolysis is desired uncoated forms may be used. Some forms mayinclude enzymes capable of cleaving the esters to release the ketonebodies sucha s those referred to in Doi. Microbial Polyesters.

Intravenous infusion of sodium salts of D-β-hydroxybutyrate has beenperformed on normal human subjects and patients for a number ofconditions, eg. those undergoing treatment for severe sepsis in anintensive care unit. It was found to be non-toxic and capable ofdecreasing glucose free fatty acids and glycerol concentration, butineffective in decreasing leucine oxidation.

The monomer of D-β-hydroxybutyrate is a white, odourless crystal with aslightly tart or acid taste which is less in intensity in comparison tovinegar or lemon juice. It can be formulated into most foodstuffs, eg.drinks, puddings, mashed vegetables or inert fillers. The acid forms ofD-β-hydroxybutyrate are suitable for use orally as they have a pKa of4.4. This is less acid than citric acid with pKal of 3.1 and pKa2 of 4.8and slightly more acidic than acetic acid with a pKa of 4.7.

Preferably, only the natural D- or (R) isomer is used in thisformulation. Since in practice it is not possible to achieve absoluteisomeric purity, the article of commerce currently sold by Sigma, St.Louis Mo. or Fluka, Ronkonkoma, N.Y. is the most suitable for thispurpose. The optical rotation of the commercially availableD-β-hydroxybutyratic acid is −25°±1 at the wavelength of Na and itsmelting point 43-46° C. The optical rotation of the Na salt ofD-β-hydroxybutyrate is −14.5° and its melting point 149-153° C. Both canbe assayed by standard enzymatic analysis using D-β-hydroxybutyratedehydrogenase (EC 1.1.1.30)(5). Acetoacetate can be determined using thesame enzyme (56). The unphysiological (S) isomer is not measurable withenzymatic analysis but can be measured using GC mass spec (13).

For a 1500 calorie diet, the human adult patient could consume 198 g ofketones per day. For a 2000 calorie diet of the same proportions, onecould consume 264 g of ketones per day. On the ketogenic lipid dietblood ketones are elevated to about 2 mM. On the ketone diet, ketonelevels should be higher because ketones have been substituted at thecaloric equivalent of fat, that is 1.5 g of ketone/1 g of fat.Accordingly, blood ketones should be approximately 3 mM, but still belowthe level achieved in fasting man of 7.5 mM.

The advantage of using ketone bodies themselves are several. Firstly,provision of ketone bodies themselves does not require the limitation ofcarbohydrate, thus increasing the palatability of the dietaryformulations, particularly in cultures where high carbohydrate diets arecommon. Secondly, ketone bodies can be metabolized by muscle, heart andbrain tissue, but not liver. Hence the fatty liver, which may be anuntoward side effect of the ketogenic diet, is avoided. Thirdly, theability to include carbohydrate in the dietary formulations increasesthe chance of compliance and opens up practical therapeutic approachesto type II diabetics where insulin is high, making the known ketogenicdiet unworkable.

The present inventor has determined that, while any elevation of ketonebodies may be desirable, a preferred amount of ketone bodies to beadministered will be sufficient to elevate blood levels to the 0.5 to 20mM level, preferably to the 2 mM to 7.5 mM level and above, particularlywhen attempting to arrest the death of brain cells in diseases such asAlzheimer's. While dead cells cannot be restored, arrest of furtherdeterioration and at least some restoration of function is to beanticipated.

Thus in a first aspect of the present invention there is provided theuse acetoacetate, D-β-hydroxybutyrate or a metabolic precursor of eitherin the manufacture of a medicament or nutritional aid (i) for increasingcardiac efficiency, particularly efficiency in use of glucose (ii) forproviding energy source, particularly in treating diabetes and insulinresistant states or by increasing the response of a body to insulin(iii) for reversing, retarding or preventing nerve cell damage or deathrelated disorders, particularly neurodegenerative disorders such asmemory associated disorders such as Alzheimer's, seizure and relatedstates such as encepalophies such as CJD and BSE.

The term metabolic precursor thereof particularly relates to compoundsthat comprise 1,3-butandiol, acetoacetyl or D-β-hydroxybutyrate moietiessuch as acetoacetyl-1,3-butandiol, acetoacetyl-D-β-hydroxybutyrate, andacetoacetylglycerol. Esters of any such compounds with monohydric,dihydric or trihydric alcohols is also envisaged.

This aspect includes such use as a neuronal stimulant eg capable ofstimulating axonal and/or dendritic growth in nerve cells, eg. inHippocampus or Substantia nigra particularly in diseases whereneurogeneration has serious clinical consequences.

In diabetic patients this use of these compounds allows maintenance oflow blood sugar levels without fear of hypoglycemic complications. Innormal non-diabetic subjects the fasting blood sugar is 80 to 90 mg %(4.4-5 mM) rising to 130 mg % (7.2 mM) after a meal. In diabetics ‘tightcontrol’ of diabetes has long been recommended as a method forretardation of vascular complications but, in practice, physicians havefound it difficult to keep blood sugars tightly controlled below 150 mg% (8.3 mM) after eating because of hypoglycaemic episodes. Hypoglycaemiccoma occurs regularly in normal subjects whose blood sugar drops to 2mM. As discussed earlier, (62, 63) in the presence of 5 mM blood ketonesthere are no neurological symptoms when blood sugars fall to below 1 mM.

The present inventor has determined that supplementing type II diabeticswith ketone bodies would allow better control of blood sugar, thuspreventing the vascular changes in eye and kidney which occur now after20 years of diabetes and which are the major cause of morbidity andmortality in diabetics.

Where the therapy is aimed at seizure related disorders, such asrefractory epilepsy as is treated by the ketogenic diet, therapy isimproved by use of ketone bodies, their polymers or esters or precursorssuch as butandiol compounds, due to the reduction or elimination of highlipid and carbohydrate content. Such patients include those with geneticdefects in the brain glucose transporter system, in glycolysis or in PDHitself such as in Leigh's syndrome.

Particular disorders treatable with these medicaments are applicable toall conditions involving PDH blockage, including those conditionsoccuring after head trauma, or involving reduction or eleimination ofacetyl CoA supply to the mitochondrion such as insulin coma andhypoglycaemia, defects in the glucose transporter in the brain or inglycolytic enzyme steps or in pyruvate transport.

Preferably the hydroxybutyrate is in the form of non-racemicD-β-hydroxybutyrate and more preferably it is administered in a formwhich also supplies acetoacetate. Preferably the metabolic precursor isone which when administered to a human or animal body is metabolised,eg. by liver, to produce one or both of D-β-hydroxybutyrate andacetoacetate, more preferably in a physiological ratio. Particularlypreferred are poly-D-β-hydroxybutyric acid oracetoacetoyl-β-hydroxybutyrate oligomers or an ester of one or both ofthese. Lower alkyl esters such as C₁₋₄ alkyl esters may be employed butmore preferably are more physiologically acceptable esters such as therespective 1,3-butandiol esters, particularly employing(R)-1,3-butandiol. Most preferred are the acetoacetyl-tri-, tetra- andpenta-D-β-hydroxybutyrate esters. Ester precursors will include estersof 1,3-butandiol, preferably (R) form and particularly acetoacetateesters such as acetoacetyl glycerol.

Preferred poly D-β-hydroxybutyrate esters are those which are esters ofthe preferred oligomers of 2-100 repeats, eg. 2-20 repeats mostpreferably 2-10 repeats.

Where the medicament or nutritional product of the invention is for usewithout prolonged storage it is convenient to use it in the form of aliquid or solid composition comprising the hydroxy substitutedcarboxylic acid and/or the ketone, preferably comprising both and wherethese are the D-β-hydroxybutyrate acids and acetoacetate togetherpreferably in the ratio of about 3:1 to 5:1, more preferably about 4:1.

Where the medicament or aid comprises acetoacetate it is preferably notstored for a prolonged period or exposed to temperatures in excess of40° C. Acetoacetate is unstable on heating and decomposes violently at100° C. into acetone and CO₂. In such circumstances it is preferred thatacetoacetate is generated by the composition on contact with the bodiesmetabolic processes. Preferably the composition comprises an esterprecursor of actetoacetate. For example, the ethyl ester of acetoacetateis relatively stable with a boiling point of 180.8° C.

Still more preferably, the medicament or aid comprises an acetoacetylester of D-β-hydroxybutyrate or such an ester of an oligomer ofD-β-hydroxybutyrate as described. This may be supplemented withD-β-hydroxybutyrate or one of the polymers of that, e.g.oligo-D-β-hydroxybutyrate, in order to bring about the preferred ratioof the two components. Such a composition will provide the two preferredcomponents when the ester and polymer are metabolised in the stomach orin the plasma of the human or animal which has consumed them. Again an(R) 1,3-butandiol ester of the acetoacetyl-D-β-hydroxybutyrate may bemost preferred as it will be more lipophilic until metabolised orotherwise deesterified and all its components are convereted to thedesired ketone bodies.

A second aspect of the invention provides novel esters of acetoacetatefor use in therapy or as a nutritional aid. Such esters may include C₁₋₄alkyl esters but most preferred are the D-β-hydroxybutyryl-acetoacetateesters referred to above.

A third aspect of the present invention provides apoly-D-β-hydroxybutyrate for use in therapy, particularly where this isin a form selected for its ability to be degraded in acid conditions ofthe stomach or by esterases in vivo.

A fourth aspect of the invention provides a method for the synthesis ofD-β-hydroxybutyryl-acetoacetate esters comprising the reaction ofacetoacetic acid halide, e.g. acetoacetyl chloride, withD-β-hydroxybutyrate. Preferably this is achieved by reacting acetoaceticacid with an activating agent, such as thionyl chloride, to produce theacid chloride.

A fifth aspect of the present invention provides a method for thesynthesis of D-β-hydroxybutyryl-acetoacetate esters comprising thereaction of D-β-halobutyrate or its oligomers with acetoacetic acid,activated forms thereof or diketene .

A sixth aspect of the present invention provides aD-β-hydroxybutyryl-acetoacetate ester per se, a physiologicallyacceptable salt or short or mdium chain mono, di or trihydric alcohol or1,3-butandiol estsr thereof.

A seventh aspect of the present invention providespoly-D-β-hydroxybutyrate together with a pharmaceutically orphysiologically acceptable carrier.

An eighth aspect of the present invention provides a compositioncomprising D-β-hydroxybutyrate and acetoacetic acid in a ratio of from1;1 to 20:1, more preferably 2:1 to 10:1 and most preferably from 3:1 to5:1 together with a pharmaceutically or physiologically acceptablecarrier. Preferably the ratio of these components is about 4:1. Such acomposition does not consist of plasma, serum or animal or plant tissuealready used as a medicament or foodstuff, thus it is that which ispreferably sterile and pyrogen free. Particularly the ketione bodiescomprise at least 5% of the composition by weight, more preferably 20%or more and most preferably 50% to 100%. The composition may be adaptedfor oral, parenteral or any other conventional form of administration.

A ninth aspect of the present invention comprises a method of treating ahuman or animal in order to increase their cardiac efficiency comprisingadministering to that person at least one of a materials for use in thefirst to eight aspects of the invention.

A tenth aspect of the present invention comprises a method of treating ahuman or animal in order to increase their the response to insulincomprising administering to that person at least one of a materials foruse in the first to eight aspects of the invention.

An eleventh aspect of the present invention comprises a method oftreating a human or animal in order to treat an insulin resistant statecomprising administering to that person least one at least one of amaterials for use in the first to eight aspects of the invention.

By insulin resistant state herein is included forms of diabetes,particularly those that do not respond fully to insulin.

A twelvth aspect of the invention provides a method of treating a humanor animal in order to treat a nerve cell, eg. brain cell, death ordamage related disorder as referred to for the first aspect,particularly a neurodegenerative disorder eg. such as those related toneurotoxic conditions such as presence of amyloid protein, eg. a memoryassociated disorder such as Alzheimer's disease, or epileptic seizures,comprising administering to that person least one at least one of amaterials for use in the first to eight aspects of the invention.

Preferred methods of the ninth to twelvth aspects of the invention usethe preferred ketones and polyacids and acid esters of the invention.

Methods of preparing poly D-β-hydroxybutyrate are not specificallyclaimed as these are known in the art. For example Shang et al, (1994)Appli. Environ. Microbiol. 60: 1198-1205. This polymer is availablecommercially from Fluka Chemical Co. P1082, cat#81329, 1993-94, 980.Second St. Ronkonkoma N.Y. 11779-7238, 800 358 5287.

Particular advantages of use of the biologically available polymers ofthe invention include the reduction in the amount of counter ions suchas sodium that have to be coadministered with them. This reduction insodium load is advantageous particularly in ill health. By biologicallyavailable is meant those materials which can be used by the body toproduce the least one of a D-β-hydroxybutyrate, acetoacetate and amixture of these in physiological ratio as described above

The amount of ketone bodies used in treatment of neurodegeneration suchas Alzheimer's and Parkinsonism will preferably elevate blood levels to0.5 mM to 20 mM, eg 2 mM to 7.5 mM as described above. The presentinventor estimates that 200 to 300 g (0.5 pounds) of ketone bodies perpatient per day mignt be required to achieve this. Where the treatmentis through maintenance of cells against the effects of neurotox in thismay be at a higher level, eg. 2 to 7.5 mM in blood. Where it relies onthe nerve stimulatory factor effect of the D-β-hydroxybutyrate soproduced the amount administered may be lower, eg. to provide 0.2 to 4mM, but can of course be more for this or other disease.

It will be realised that treatment for neurodegenerative diseases suchas Alzheimer's will most effectively be given soon after identifyingpatient's with a predisposition to develop the disease. Thus treatmentfor Alzheimers' most effectively follows a positive test result for oneor more conditions selected from the group (i) mutations in the amyloidprecursor protein gene on chromosome 21, (ii) mutations in thepresenilin gene on chromosome 14, (iii) presence of isoforms ofapolipoprotein E. Other tests shown to be indicative of Alzheimer's willof course be applicable.

Following such a positive test result it will be appropriate to preventthe development of memory loss and/or other neurological dysfunction byelevation of the total sum of the concentrations of the ketone bodiesD-β-hydroxybutyrate and acetoacetate in the patient's blood or plasma tosay between 1.5 and 10 mM, more preferably 2 to 8 mM, by one of severalmeans. Preferably the patient is fed a diet of sufficient quantities ofD-β-hydroxybutyrate, its metabolisable polymers, its acetoacetate estersor their precursors (R)-1,3-butandiol and its acetoacetate esters, eg.acetoacetyl glycerol, or its administered intravenously orintrarterially the ketone bodies D-β-hydroxybutyrate and acetoaceticacid. All of the organic materials referred to above are optionally insalt or ester form. Examples of typical physiologically acceptable saltswill be selected from sodium, potassium, magnesium, L-Lysine andL-arginine or eg. more complex salts such as those of methyl glucaminesalts. Esters will be those as described previously for other aspects ofthe invention.

A still further aspect of the invention provides the ketone bodies ofthe invention by suitable control of diet. Thus this aspect provides amethod of treatment of a human or animal for a disorder of one or moreof the ninth to the twelvth aspects of the invention comprising one of(i) total fasting of the individual and (ii) feeding the individual aketogenic diet eg. of 60-80% lipid with carbohydrate content 20% or lessby weight.

For the purpose of treaing seizures, eg. in epilepsy, a diet may involvead lib ingestion of carbohydrate by oral or enteral route or of thecompounds specified above.

In all these treatments other than the ketogenic diet there is theimprovement that a method of avoiding drop in blood ketones whichaccompanies the ingestion of excess carbohydrate and a method whichavoids feeding of excess lipid which accelerates the synthesis by liverof fatty acids and cholesterol which would otherwise contribute tovascular disease.

It will be realised that hypoglycemic brain dysfunction will also betreatable using the treatments and compositions and compounds of thepresent invention. A further property associated with the presenttreatment will be general improvement in muscle performance.

The provision of ketone body based foodstuffs and medicaments of theinvention is faciliated by the ready availability of a number ofrelatively cheap, or potentially cheap, starting materials from which(R)-3-hydroxybutyric acid may be derived (see Microbial PolyestersYoshiharu Doi. ISBN 0-89573-746-9 Chapters 1.1, 3.2 and 8). Theavailability of genes capable of insertion into foodstuff generatingorganisms provides a means for generating products such as yoghurts andcheese that are enriched in either poly-(R)-3-hydroxybutyric acid or,after breakdown with enzymes capable of cleaving such polymers, with themonomeric substance itself (see Doi. Chapter 8).

The present invention will now be described further by way ofillustration only by reference to the following Figures and experimentalexamples. Further embodiments falling within the scope of the inventionwill occur to those skilled in the art in the light of these.

FIGURES

FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate level producedafter time after gavage of (R)-3-hydroxybutyrate, an oligomer of this asproduced in Example 1 and an acetoacetyl monomer therof as produced inExample 2;

FIG. 2 is a graph showing blood (R)-3-hydroxybutyrate level producedafter time after feeding rats with the triolide of(R)-3-hydroxybutyrate, a cyclic oligomer produced in Example 1 inyoghurt and controls fed yoghurt alone; and

FIG. 3 shows the reactions of the Krebs cycle.

EXAMPLES Example 1 Preparation of Oligomers of (R)-3-Hydroxybutyric Acid(D-β-Hydroxybutyrate)

(R)-3-hydroxybutyric acid (Fluka-5.0 g: 0.048 mole), p-toluene sulphonicacid (0.025 g) and benzene (100 ml) were stirred under reflux withn aDean-Stark trap arrangement for 24 hours. The reaction mixture wascooled and the benzene evaporated in vacuo (0.5 mm Hg). 4.4 g ofcolourless oil was obtained of which a 20 mg sample was converted to themethyl ester for analysis of number of monomer repeats using NMR. Thesestudies show that the product is a mixture of oligomers ofD-β-hydroxybutyrate of avaerage number of repeats 3.75, being mainly amixture of trimers, tetramers and pentamers with the single mostabundant material being the tetramer. The product mixture was soluble in1 equivalent of sodium hydroxide.

Example 2 Preparation of Acetoacetyl Ester of Oligomeric(R)-3-Hydroxybutyric Acid

A further batch of the colourless oil product from Example 1 (4.5 g) washeated for 1 hour at 60° C. with diketene (3.8 g) and sodium actetate(0.045 g) under nitrogen. Further diketene (3.8 g) was added and thereaction heated for a further hour, cooled and diluted with ether,washed with water and then extracted with saturated sodium bicarbonate(5×100 ml). Combined extract was washed with ether then acidified withconcentrated HCl (added dropwise). Ethyl acetate extraction (3×50 ml)was followed by drying over magnesium sulphate and evaporation in vacuo.A yellow solid/oil mixture was obtained (7.6 g) which waschromatographed on a silica column using dichloromethane/methanol (98:2)to give a light amber oil product. Faster moving impurities wereisolated (1.6 g) and after recolumning carbontetrachloride/methanol(99:1) 0.8 g of oil was recovered which was shown by NMR and Massspectrometry to be the desired mixture of acetoacetylated oligomers ofD-β-hydroxybutyrate. The product mixture had an Rf of 0.44 indichloromethane/methanol (90:1) and was soluble in 1 equivalent ofsodium hydroxide. Both products of Example 1 and Example 2 aresusceptible to separation of individual components by preparative HPLC.

Example 3 Oral Administration of D-β-Hydroxybutyrate, Oligomers andAcetoacetyl D-β-Hydroxybutyrate Oligomers to Rats

The ability of orally administered D-β-hydroxybutyrate and the oligomersof Examples 1 and 2 to raise blood ketone body levels was investigatedas follows. Rats were starved overnight and then gavaged with 100 μl/100g bodyweight of 4M D-β-hydroxybutyrate brought to pH 7.74 using methylglucamine. Blood levels of D-β-hydroxybutyrate measured using andNAD+/EDTA assay of Anal. Biochem. 131, p 478-482 (1983). 1.0 ml of asolution made up from 2-amino-2-methyl-1-propanol (100 mM pH 9.9, 0.094g/10 ml), NAD+ (30 mM, 0.199 g/10 ml) and EDTA (4 mM, 0.015 g/10 ml) wasadded to each of a number of cuvettes and 4 μl sample orD-β-hydroxybutyrate control.

As the rats had been fasted the initial levels of D-β-hydroxybutyratewere elevated from the 0.1 mM fed state. However, consistent serumincreases of D-β-hydroxybutyrate, between 1 and 3.2 mM increase in eachcase, were provided.

This procedure was repeated with 2M solutions of the mixtures ofD-β-hydroxybutyrate oligomers and their acetoacetyl esters described inExamples 1 and 2. The D-β-hydroxybutyrate oligomer (19/1 in FIG. 1) andthe acetoacetyl ester (20/4 in FIG. 1) were both brought to pH 7.6 withmethyl glucamine and the blood D-β-hydroxybutyrate level monitored usingthe aforesaid assay procedure. Increases in serum D-β-hydroxybutyratewere shown to be of 0.5 to 1.2 mM at 60 and 120 minutes after gavaging.These results demonstrate the efficacy of orally administeredD-β-hydroxybutyrate and its metabolic precursors of the invention inraising blood levels significantly for a period of hours after intake.

It was noted that the oligomeric esters 19/1 and 20/4, while notelevating the blood ketone body level as high as the monomer itself, didresult in elevation for a much longer period of time and thus are suitedto adminsitration less frequently than the monomer.

Example 4

TABLE 2 Sample 1500 calorie ketogenic diet using ketone bodies, theiresters or polymers. The ketones were assumed to contain 6 kcal/g, fats 9kcal/g, carbohydrate and protein 4 kcal/g. Ketones have been substitutedto give equivalent calories. Amount Fat Protein CHO Ketones (g) (g) (g)(g) (g) Breakfast Egg 32 4 4 apple juice 70 7 ketones 66 66 skim milk 920 2 3 Total Breakfast 4 6 10 66 Lunch Lean beef 12 1.75 3.5 cookedcarrots 45 0.6 3 canned pears 40 4 ketones 69.75 69.75 skim milk 92 2 3Total Lunch 1.75 6.1 10 69.75 Supper Frankfurter 22.5 6 3 cookedbroccoli 50 1 2 watermelon 75 5 ketones 62.25 62.25 skim milk 92 2 3Total Supper 6 6 10 62.25 Daily Total 11.75 18.1 30 198

Example 5 Effect of Increased Blood D-β-Hydroxybutyrate Levels on WholeBrain GABA Levels

To assess the effect of D-β-hydroxybutyrate on whole brain GABA levels,and thus provide an indication of antiepileptic effect of ketone body orprecursor treatment aimed at increasing blood ketone body levels, wholerat brain was frozen at set times after administration ofD-β-hydroxybutyrate as described in Example 3. GABA was assayed usingstandard HPLC technique and related to protein content using standardprotein assay. At t=0 GABA levels were 191 pmoles/μg protein while at120 minutes this was elevated at 466 pmoles/μgprotein, demonstratingantepileptic potential.

Example 6 Effect of D-β-Hydroxybutyrate on β-Amyloid Toxicity toHippocampal Cells in Vitro Culture Medium and Chemicals

The serum free medium used from 0 to day 4 contained Neurobasal mediumwith B27 supplement diluted 50 fold (Life Technology, Gaithersburg, Md.)to which was added: 0.5 mM L-glutamine, 25 μM Na L-glutamate, 100 U/mlpenicillin and 100 μg/ml streptomycin. After day 4, DMEM/F12 mediumcontaining 5 μM insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Naselenite 100 U/ml penicillin and 100 μg/ml streptomycin were used.

Hippocampal Microisland Cultures

The primary hippocampal cultures were removed from Wistar embryos on day18 and dispersed by gentle aggitation in a pipette. The suspension wascentrifuge at 1,500×g for 10 min and the supernatant discarded. Newmedia was make 0.4-0.5×10⁶ cells/ml. Ten μl of this suspension waspipetted into the center of poly D-lysine coated culture wells and theplates incubated at 38° C. for 4 hrs and then 400 μl of fresh

Neurobasal media was added. After 2 days of incubation, half of themedia was exchanged for fresh media and the incubation continued for 2more days. After day 4, the medium was changed with DMEM/F12 mediumcontaining 5 μM insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Naselenite 100 U/ml penicillin and 100 μg/ml streptomycin. The wells weredivided into 4 groups: half the wells received Na D-β-hydroxybutyrate toa final concentration of 8 mM while and half of the wells received 5 nMamyloid β₁₋₄₂ (Sigma). These media were exchanged 2 days later (day 8)and the cells were fixed on day 10 and stained with anti MAP2(Boehringer Manheim, Indianapolis Ind.) to visual neurons and vimentinand GFAP (Boehringer) to visualize glial cells.

Results Cell Counts

Addition of D-β-hydroxybutyrate to the incubation resulted in anincrease in the neuronal cell number per microisland from a mean of 30to mean of 70 cells per microisland. Addition of 5 nM amyloid β₁₋₄₂ tothe cultures reduced the cell numbers from 70 to 30 cells permicroisland, confirming the previous observations of Hoshi et al, thatamyloid β₁₋₄₂ is toxic to hippocampal neurons. AdditionD-β-hydroxybutyrate to cultures containing amyloid β₁₋₄₂ increased thecell number from a mean of 30 to 70 cells per microisland. From thesedata we conclude that addition of substrate level quantities ofD-β-hydroxybutyrate, to media whose major nutrients are glucose,pyruvate and L-glutamine, slows the rate of cell death in culture. Wefurther conclude that D-β-hydroxybutyrate can decrease the increasedrate of hippocampal cell death caused by the addition of amyloid β₁₋₄₂in culture.

The number of dendritic outgrowths and the length of axons were bothobserved to have increased with presence of D-β-hydroxybutyrate, whetherβ₁₋₄₂ was present or not. This is indicative of nerve growth factor likebehaviour.

Example 7 Preparation of(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione: triolide of(R)-3-hydroxybutyric acid

Synthesis was as described in Angew. Chem. Int. Ed. Engl. (1992), 31,434. A mixture of poly[(R)-3-hydroxybutyric acid] (50 g) andtoluene-4-sulphonic acid monohydrate (21.5 g, 0.113 mole) in toluene(840 ml) and 1,2-dichloroethane (210 ml) was stirred and heated toreflux for 20 hours. The water was removed by Dean-stark trap for 15hours whereafter the brown solution was cooled to room temperature andwashed first with a half saturated solution of sodium carbonate thenwith saturated sodium chloride, dried over magnesium sulphate andevacuated in vacuo. The brown semi-solid residue was distilled using aKugelrohr apparatus to yield a white solid (18.1 g) at 120-130° C./0.15mmHg. Above 130° C. a waxy solid began to distill—distillation beingstopped at this point. The distilled material had mp 100-102° C.(literature mp 110-110.5° C.). Recrystallisation from hexane gavecolourless crystals in yield 15.3 g. Mp=107-108° C.; [α]_(D)-35.1(c=1.005, CHCl₃), (lit.=−33.9). ¹H NMR (300 MHz, CDCl₃): δ=1.30 (d, 9H,CH₃); 2.4-2.6 (m,6H; CH₂); 5.31-5.39 (M, 3H; HC—O). ¹³C NMR (CDCl₃)δ=20.86 (CH₃), 42.21 (CH₂), 68.92 (CH), 170.12 (CO). Elemental analysis:calculated for C₁₂H₁₈O₆: C, 55.81; H, 7.02; Found: C, 55.67; H, 7.15.

Example 8 Oral Administration of Triolide of D-β-Hydroxybutyrate ofExample 1 to Rats

The ability of orally administered triolide to raise blood ketone levelswas investigated as follows. The day before the experiment commenced, 12Wistar rats weighing 316±10 g were placed in separate cages. They had noaccess to food for 15 hours prior to presentation with triolidecontaining compositions, but water was provided ad libitum.

On the morning of the experiment 0.64 g of triolide was mixed with 5 gCo-op brand Black Cherry yoghurt in separate feeding bowls for 9 of therats. The remaining 3 rats were given 5 g of the yoghurt without thetriolide as controls. The yoghurt containing bowls were placed in thecages and the rats timed while they ate. Two of the three control ratsate all the yoghurt and four of the six triolide yoghurt rats ateapproximately half the provided amount. The remaining six rats slept.

Control rats (n=2) were killed at 60 and 180 minutes after ingestion ofyoghurt while triolide fed rats were killed at 80, 140, 150 and 155minutes. Blood samples were taken for assay of D-β-hydroxybutyrate.Brains were funnel frozen and later extracted in perchloric acid andextracts neutralised and assayed. Blood levels of (R)-3-hydroxybutyratewere measured using a NAD⁺/EDTA assay of Anal. Biochem (1983) 131, p478-482. 1.0 ml of a solution made up from 2-amino-2-methyl-1-propanol(100 mM pH 9.9, 0.094 g/10 ml), NAD⁺ (30 mM, 0.199 g/10 ml) and EDTA (4mM, 0.015 g/10 ml) was added to each of a number of cuvettes and 4 μlsample or D-β-hydroxybutyrate control.

The two control rats ate 5.2±0.1 g yoghurt and their plasma(R)-3-hydroxybutyrate concentrations were about 0.45 mM at 60 minutesand 180 minutes. The four triolide fed rats ate 0.39±0.03 g of thetriolide and 2.6±0.2 g of yoghurt. Their plasma D-β-hydroxybutyrateconcentrations were 0.8 mM after 80 minutes and 1.1 mM for the groupsacrificed at about 150 minutes. All rats displayed no ill effects fromingestion of triolide.

The test rats thus showed increase in plasma D-β-hydroxybutyrate over atleast 3 hours with no ill effects. It should be noted that two otherrats fed approximately 1.5 g triolide each in ‘Hob-Nob’ biscuit showedno ill effects after two weeks.

For all examples given above, it should be noted that the increasedlevels of (R)-3-hydroxybutyrate will also be mirrored in acetoacetatelevels, not measured here, as there is a rapid establishment ofequilibrium between the two in vivo such that acetoacetate levels willbe between 40 and 100% of the (R)-3-hydroxybutyrate levels.

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1. A method of a treating a patient for a neuro-degenerative disorder comprising administering to that patient a therapeutically effective amount of one or more of D-β-hydroxybutyric acid, acetoacetate, or a metabolic precursor or physiologically acceptable salt of D-β-hydroxybutyric acid or acetoacetate, such as to elevate the patient's blood level of ketone bodies, defined as the sum total of D-β-hydroxyburyric acid and acetoacetate, to a therapeutic level effective to treat the disorder wherein when a metabolic precursor is administered it is not hydroxybutyryl carnitine.
 2. A method of treating a patient in order to treat a neuro-degenerative disorder comprising administering to that patient a therapeutically effective amount of at least one of D-β-hydroxybutyric acid, acetoacetate, or a metabolic precursor or physiologically acceptable salt of D-β-hydroxybutyric acid or acetoacetate, such as to elevate the patient's blood level of ketone bodies, defined as the sum total of D-β-hydroxybutyric acid and acetoacetate, to a therapeutic level effective to treat the disorder wherein the patient's blood level is elevated to from 0.3 mM to 20 mM.
 3. A method of treating a CNS cell, peripheral nerve cell, or otherwise insulin insensitive cell in need of therapy for one or more of neuro-degeneration, GABA preventable seizure, or insufficient ability to metabolise glucose, comprising administering to that cell one or more compounds selected from the group consisting of D-β-hydroxybutyric acid, acetoacetate, compounds which are oligomers of D-β-hydroxybutyric acid, acetoacetyl esters of D-β-hydroxybutyric acid and acetoacetyl esters of oligomers of D-β-hydroxybutyric acid, and physiologically acceptable salts thereof.
 4. A method of treating an patient for epilepsy, diabetes or an insulin resistant state comprising administering to that patient a therapeutically effective amount of one or more compounds selected from the group consisting of D-β-hydroxybutyric acid, acetoacetate and metabolic precursors of D-β-hydroxybutyric acid or acetoacetate which comprise moieties selected from the group consisting of R-1,3-butandiol, acetoacetyl and D-β-hydroxybutyryl moieties and physiologically acceptable salts and esters thereof.
 5. A method as claimed in claim 1 wherein on administration of the compound to an unfasted patient in need of such therapy, the blood level of ketone bodies, defined as the sum total of D-3-hydroxybutyric acid and acetoacetate, is raised to between 0.3 and 20 mM.
 6. A method as claimed in claim 1 wherein the neurodegenerative disorder is selected from the group consisting of neurodegenerative disorders involving inability to metabolise glucose, memory loss in ageing, neurotoxic peptides or proteins, and genetic abnormality.
 7. A method as claimed in claim 6 wherein the neurodegenerative disorder is selected from those involving neurotoxic protein plaques.
 8. A method as claimed in claim 1 wherein the metabolic precursor is selected from the group consisting of Free Fatty Acids and compounds comprising 1,3-butandiol, acetoacetyl or D-β-hydroxybutyryl moieties.
 9. A method as claimed in claim 1, wherein the metabolic precursor is a polymer or oligomer of D-β-hydroxybutyrate.
 10. A method as claimed in claim 9 wherein the metabolic precursor is an aceroacetyl ester.
 11. A method as claimed in claim 9 wherein metabolic precursor is selected from the group consisting of compounds of general formulae

or physiologicial acceptable salts or esters thereof wherein in each case n is selected such that the polymer or oligomer is readily metabolised on administration to a human or animal body to provide elevated ketone body levels in blood.
 12. A method as claimed in claim 11 wherein n is an integer of 0 to 1,000.
 13. A method as claimed in claim 11 wherein n is an integer of from 1 to
 5. 14. A method as claimed in claim 1, wherein the level of ketone bodies produced in the blood is in the ratio 1:1 to 20:1 of D-β-hydroxybutyrate to acetoacetate.
 15. A method as claimed in claim 9 wherein the oligomer is a cyclic oligorner of formula

where n is an integer of 1 or more or a complex thereof with one or more cations or a salt thereof
 16. A method as claimed in claim 15 wherein the one or more cations are selected from the group consisting of sodium, potassium, magnesium and calcium.
 17. A method as claimed in claim 15 wherein n is an integer from 1 to
 20. 18. A method as claimed in claim 1 wherein it is (R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione.
 19. A compound of formula

or physiologicial acceptable salts or esters thereof. wherein n is an integer from 0 to 1000
 20. A compound as defined in claim 19 wherein the ester is selected from the group consisting of monohydric, dihydric or trihydric alcohol esters.
 21. A compound as claimed in claim 19 wherein the ester is of (R)-1,3-butandiol.
 22. A compound as claimed in claim 19 wherein n is selected from the group of integers 0, 1, 2, 3 and
 4. 23. A foodstuff comprising poly D-β-hydroxybutyrate characterised in that it is derived from a foodstuff generating organism that has had a gene capable of producing D-β-hydroxybutyrate inserted therein.
 24. A foodstuff characterised in that it comprises at least 5% ketone bodies by weight.
 25. A method for the synthesis of D-β-hydroxybutyryl-acetoacetate or poly or oligo-D-β-hydroxybutyryl-acetoacetate esters comprising the reaction of acetoacetic acid halide with D-β-hydroxybutyrate or poly- or oligo-D-β-hydroxybutyrate.
 26. A method for synthesis of D-β-hydroxybutyryl-acetoacetate or oligo-D-β-hydroxybutyryl-acetoacetate comprising reacting D-β-hydroxybutyryic acid with diketene.
 27. A method of synthesising an oligomer of D-β-hydroxybutyric acid comprising heating a solution of D-β-hydroxybutyric acid in a solvent until an oligomer of a desired number of repeats is produced.
 28. Use of D-β-hydroxybutyric acid, acetoacetate, or a metabolic precursor or physiologically acceptable salt of D-β-hydroxybutyric acid or acetoacetate for the manufacture of a medicament for the treatment of a disorder by a method as set out in claim 1 provided that when the use is of a metabolic precursor that is not racemic hydroxybutyryl carnitine.
 29. A foodstuff as claimed in claim 23 for use in therapy.
 30. Poly-D-β-hydroxybutyrate for use in therapy.
 31. A composition comprising a compound selected from those claimed in claim 15 and poly D-β-hydroxybutyrate together with a physiologically acceptable carrier, in sterile and pyrogen free form. 